Method for determination of antithrombin III activity and reagent kit therefor

ABSTRACT

PCT No. PCT/JP94/01024 Sec. 371 Date Feb. 17, 1995 Sec. 102(e) Date Feb. 17, 1995 PCT Filed Jun. 27, 1994 PCT Pub. No. WO95/00663 PCT Pub. Date Jan. 5, 1995An improved method of a conventional antithrombin III activity determination method, which does not require dilution of a sample and can avoid influence of heparin cofactor II is provided. This method is characterized in that the reaction of a sample with thrombin in the presence of heparin is carried out in the presence of more than 0.2 to 0.9M of a salt, while such a reaction is carried out in the presence of 0.2M of a salt in a conventional method.

INDUSTRIAL FIELD OF APPLICATION

The present invention relates to a method for the determination ofantithrombin III (hereinafter abbreviated as ATIII) activity in bodyfluids. More specifically, it relates to an improved method for thedetermination of ATIII activity in body fluids comprising reacting asample with thrombin in the presence of heparin and a salt and thenmeasuring a color developed from a chromogenic substrate to determinethe remaining thrombin activity. The present invention further relatesto a reagent kit to be used in the method for the determination of ATIIIactivity.

PRIOR ART AND PROBLEMS

ATIII is a serine protease inhibitor present in blood in a large amount(20 to 30 mg/dl) and is known as a blood coagulation inhibitor. Bloodcoagulation takes place by an amplification reaction due to cascade-likefunction of many proteases. In the final stage of blood coagulation,thrombin thus formed converts fibrinogen into fibrin and, in addition,thrombin activates blood coagulation factor XIII. The activated bloodcoagulation factor XIII forms crosslinkages between fibrins to formthrombi. A most important regulator of this formation of thrombi isATIII and ATIII binds to thrombin and proteases which participate inblood coagulation such as blood coagulation factor X and the like toinhibit them. When ATIII in blood is lowered, thrombi are liable to beformed. Therefore, lowering of ATIII in blood causes trouble. It hasbeen known that lowering of ATIII in blood occurs by, for example, lownutriture, severe hepatic diseases, coagulation sthenia such as diffuseintravascular coagulation (DIC), acute thrombosis and the like,nephrotic syndrome, and congenital ATIII deficiency or abnormality.Then, ATIII is an important indication in clinical diagnosis.

As methods for the determination of ATIII, immunological methods for thedetermination of immunizing doses, methods using fibrinogen orchromogenic substrates, and methods using ATIII deficient plasmacontaining coagulation factors participating in extrinsic reactions havealready been known. We have invented a method using ATIII deficientplasma containing coagulation factors participating in extrinsicreactions (PCT/JP89/00173 (Nov. 2, 1989); and Thrombosis Research, Vol.57, 729-736 (1990)). In this method, ATIII can be simply, easily andaccurately determined by measuring a blood coagulation time with minimuminfluence of heparin cofactor II (hereinafter abbreviated as HCII).However, since a blood coagulation time is measured in this method, itis difficult to apply the method to general automatic analyzers.

At present, methods using chromogenic substrates are widely used becausethey are readily applicable to automatic analyzers.

In a method using a chromogenic substrate, an excess amount of thrombinis added to a sample to react ATIII in the sample with thrombin in thepresence of heparin and a salt and then a chromogenic substrate is addedto the reaction mixture to measure a color developed to determine theremaining thrombin activity, that is, to indirectly determine ATIII inthe sample. This determination of ATIII is described by, for example,Rinsho Byori, Special Vol. 70, 173-177 (1987); U. Abildgaard, et al.,Thrombosis Research, Vol. 11, 549-553 (1977).

In this method for the determination of ATIII with a chromogenicsubstrate, there are two major problems. One problem is dilution of asample. In general, it is necessary to dilute a sample so as to avoidinfluence of its inherent color and turbidity upon measurement. Inaddition, since a sample contains a large amount of ATIII, when thesample is used without any dilution, a very large amount of thrombinmust be added because the amount of thrombin should exceed the amount ofATIII. In such a case, since 0% of ATIII activity corresponds to theactivity of thrombin added for preparing a calibration curve, a colorreleased from a chromogenic substrate reaches a measurable limit ofabsorbance within a very short period of time. For applying to variousautomatic analyzers, it is undesirable to exceed 2.0 of absorbancewithin an extremely short period of time, e.g., within one minute.Although influence of an inherent color and turbidity of a sample uponmeasurement can be avoided by carrying out a kinetic analysis, theproblem that the color released reaches a measurable limit of absorbancewithin a very short time still remains. Dilution of samples are veryinconvenient for routine tests. For solving this problem, it has beenproposed to use an oligopeptide substrate which has a 1/5 to 1/100 timeslower sensitivity to thrombin in comparison with Tos-Gly-Pro-Arg-pNA asa substrate in the presence of a denaturant or tetrapeptide,Gly-Pro-Arg-Pro (U.S. Pat. No. 5,320,945) (Jul. 14, 1994)

Another problem is to avoid the influence of HCII because ATIIIdetermination is influenced by HCII. HCII is present in an amount ofabout 9 mg/dl in normal plasma and is about 1/3 of ATIII. Although HCIIinhibits thrombin as ATIII does, unlike ATIII, HCII does not inhibitother coagulation factors. Like ATIII, antithrombin activity of HCII isremarkably enhanced by addition of heparin. However, it has been knownthat the optimum concentration of heparin is 1 to 5 U/ml and this ishigher than that for ATIII (0.1 to 1 U/ml) and, in addition, activationmanners by other sulfated polysaccharides are different from each other(Rinsho Byori, Special Vol. 70, 181-186 (1987); and Douglas M. Tollersenet al., The Journal of Biological Chemistry, Vol. 258, No. 11, 6713-6716(1983)). In addition, regarding affinity for heparin, HCII is weakerthan ATIII. For example, after bonding to a heparin-Sepharose column,HCII can be eluted with 0.12 to 0.16M sodium chloride, while ATIII isfirst eluted with 0.4M or more sodium chloride (Douglas M. Tollefsen etal., The Journal of Biological Chemistry, Vol. 257, No. 5, 2162-2169(1982)). In connection with this heparin affinity, the rate ofinhibition of thrombin by HCII in the presence of heparin has been knownto depend on ionic strength (Frank C. Church et al., The Journal ofBiological Chemistry, Vol. 264, No. 6, 3618-3623 (1989)). Regarding theinfluence of HCII on the determination of ATIII, there is described thatthe influence of HCII can be avoided by using bovine thrombin instead ofhuman thrombin (Jacqueline Conard et al., Thrombosis Research, Vol. 41,873-878 (1986)).

However, according to the method for determination of ATIII described inRinsho Byori, Special Vol. 70, 173-177 (1987) and U. Abildgaard et al.,Thrombosis Research, Vol. 11, 549-553 (1977), we have confirmed that,even if bovine thrombin is used, HCII greatly influences at the saltconcentration of 0.2M which is usually employed in a known method and ishigher than the salt concentration at which, according to the aboveliterature, HCII loses affinity for heparin.

As described above, since known methods for the determination of ATIIIactivity have the problem that it is influenced by HCII, an object ofthe present invention is to solve this problem. In addition, upondetermination of ATIII, a sample should be diluted. If a sample is notdiluted, a large excess amount of thrombin should be used, which resultsin the problem that an absorbance exceeds a measurable limit within avery short period of time. Another object of the present invention istherefore to solve this problem so that the determination is readilyapplicable to an automatic analyzer and thereby a simple, easy andaccurate determination method and a reagent can be provided.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 is a graph showing a calibration curve of ATIII activity at thesalt concentration of 0.10 to 0.50M according to the method of U.S. Pat.No. 4,918,001 (Apr. 17, 1990) described in Reference Examplehereinafter.

FIG. 2 is a graph showing a calibration curve of ATIII activity at thesalt concentration of 0.20 to 0.60M in a thrombin reagent (heparinconcentration: 3 U/ml) described in Example 3 hereinafter.

FIG. 3 is a graph showing the relationship between salt concentrationsand a calibration curve of ATIII at heparin concentration of 1,000 U/mldescribed in Example 4 hereinafter.

MEANS FOR SOLVING THE PROBLEMS

The present inventors have studied intensively to solve theabove-described problems. As a result, it has been unexpectedly foundthat the above problems can be solved by using a higher concentration ofa salt in a reaction mixture in a method for the determination of ATIIIactivity comprising adding thrombin to a sample, allowing to react themin the presence of heparin and a salt, and then, after the reaction,measuring a remaining thrombin activity by using a chromogenicsubstrate. Thus, the present invention has been completed.

That is, according to the present invention, there is provided a methodfor the determination of antithrombin III activity which comprisesadding a given excess amount of thrombin to a sample, reacting them inthe presence of heparin and a salt, and then measuring a remainingthrombin activity with a chromogenic substrate which develops a color bythe reaction with thrombin, the concentration of said salt being morethan 0.20M to not more than 0.90M, preferably, not less than 0.25M to0.60M, more preferably, not less than 0.35M to 0.60M.

In a known method, a salt concentration in the reaction of a sample withthrombin in the presence of heparin is 0.2M. However, in the presentinvention, the influence of HCII can be avoided by such a very simpleand less expensive means as the increase in the salt concentration. Inaddition, in the present invention, the amount of thrombin to be usedcan be reduced and thereby dilution of a sample is not required. Thus,the present invention provides such advantage that ATIII can be simply,easily and accurately determined, without dilution of a sample andexceeding a measurable limit within a short period of time, and themethod is readily applicable to automatic analyzers.

Namely, firstly, as the salt concentration in the reaction mixture of asample and thrombin increased, the influence of HCII decreased. Morespecifically, about 40% of the influence was avoided at the saltconcentration of 0.22M, about 60% of the influence was avoided at thesalt concentration of 0.24M, about 90% of the influence was avoided atthe salt concentration of 0.26M and no influence was observed at thesalt concentration of not less than 0.3M (Example 2). Then, in thedetermination of ATIII with thrombin having a certain constant activityand a given amount of a sample (Example 3), up to the salt concentrationof 0.3M, about 90% of thrombin was inhibited at 100% of ATIII activityand the linearity of a calibration curve was maintained up to 75% ofATIII activity, while, as to the salt concentration of 0.4M, about 50%of thrombin was inhibited at 100% of ATIII activity and the iinearity ofcalibration curve was maintained up to 100% of ATIII activity. As to thesalt concentration of 0.5M, about 15% of thrombin was inhibited at 100%of ATIII activity. This suggests that more amount of a sample can betested and/or thrombin activity to be added as the reagent can befurther reduced. By decreasing the amount of thrombin to be added as thereagent, another problem that the absorbance exceeds a measurable limitwithin a short period of time can be solved.

Furthermore, in the calibration curve (FIG. 2) prepared underdetermination conditions of Example 3 (heparin concentration in thereaction mixture: 3 U/ml), although the sensitivity at the saltconcentration of not less than 0.5M is not preferred, it has been foundthat the sensitivity can be improved by increasing the amount of heparin(Example 4, FIG. 3).

Thus, it has been found that the preferred sensitivity can be obtainedwithout dilution of a sample and exceeding a measurable limit ofabsorbance within a short period of time by suitably selecting theamount of a sample, thrombin activity, the salt concentration and theamount of heparin. At the same time, the influence of HCII can beavoided. In the present invention, the reaction time course is linear,the calibration curve maintains good linearity, and reproducibility isgood. Therefore, the method of the present invention is suitable forusing as routine tests.

When the method of the present invention is compared to conventionalmethods, U.S. Pat. No. 5,320,945 (Jul. 14, 1994) merely proposes achromogenic substrate which has a low sensitivity to thrombin and thereis no description of a salt concentration. No consideration is given foravoiding the influence of HCII which is one of major problems in thedetermination of ATIII. In this respect, the proposal is different fromthe method of the present invention.

U.S. Pat. No. 4,918,001 (Apr. 17, 1990) proposes a method for thedetermination of protease inhibitors, wherein a sample and a substrateare mixed, a protease is added to the mixture to start their reactionand then a hydrolyzing rate of the substrate is measured. In thismethod, ATIII is also determined and the addition of a neutral salt,preferably, sodium chloride in a concentration of 0 to 0.5M, preferably,0.08 to 0.15M is described. This method is different from conventionalmethods for the determination of ATIII in that the substrate is addedprior to addition of thrombin and no pre-heating and pre-dilution of asample are required.

In fact, when the determination of ATIII was carried out at the saltconcentration in a thrombin reagent of 0.1 to 0.5M according to themethod as Example 1 of U.S. Pat. No. 4,918,001 (Apr. 17, 1990), thecalibration curve was prepared (FIG. 1) as shown in Reference Example.However, the sensitivity is not good at the salt concentration of 0.5Mand, as to the salt concentration of 0.1 to 0.4M, the calibration curveis not suitable for routine tests because it is a curved line. Thereason why the curved line is not suitable for routine tests is that,although it is convenient for routine tests to prepare a calibrationcurve by taking only 2 points, for example, at 0 and 100% of ATIIIactivities, many further points are required for preparing a calibrationcurve in the case of the curved line as U.S. Pat. No. 4,918,001 (Apr.17, 1990). In addition, in U.S. Pat. No. 4,918,001 (Apr. 17, 1990),since the reaction time course is not linear as seen from FIG. 1 of U.S.Pat. No. 4,918,001 (Apr. 17, 1990), only limited automatic analyzers canbe used. For example, Hitachi 7150 cannot be used. Average CV ofsimultaneous reproducibilities using normal specimens and abnormalspecimens at respective salt concentrations of 0.2M, 0.3M and 0.4M (n=5)is 7.7% (Reference Example) and this is a problem for applying themethod to routine tests in accuracy. Because of these reasons, themethod of U.S. Pat. No. 4,918,001 (Apr. 17, 1990) is not desired for aroutine determination method of ATIII.

In addition, there is no suggestion of avoidance of the influence ofHCII in U.S. Pat. No. 4,918,001 (Apr. 17, 1990). Furthermore, in thepresent invention, it is essential to react a sample with thrombin inadvance and the order of reaction is of importance. In view of these,the method disclosed in U.S. Pat. No. 4,918,001 (Apr. 17, 1990) isdifferent from the method of the present invention.

Samples, reagents and the like used in the determination method of thepresent invention will be illustrated hereinbelow.

Firstly, samples to be used in the determination method of the presentinvention are, usually, plasma. Blood is collected from a subject andplasma is separated therefrom according to a conventional method.

In the determination method of the present invention, thrombin derivedfrom human being, cattle, horse, goat and the like can be used.

As chromogenic substrates which develop color by a recation withthrombin, S-2238 (H-D-Phe-Pip-Arg-pNA), Spectrozyme-TH(H-D-CHT-Ala-Arg-pNA), Chromozym-TH (Tos-Gly-Pro-Arg-pNA), CBS-34-47(H-D-CHG-But-Arg-pNA), 2AcOH-H-D-HHT-Ala-Arg-pNA and the like have beenknown and any of these thrombin substrates can be used.

As salts, neutral salts such as sodium chloride, potassium chloride andthe like are preferred. These reagents including heparin arecommercially available. Heparin and a salt may be separated from athrombin reagent. However, it is desired to incorporate them with athrombin reagent. If necessary, pH's of a thrombin reagent and achromogenic substrate reagent are adjusted. As a buffer for adjustingpH, there are tris, phosphate, barbital, imidazole, veronal,glycylglycine, BES, MOPS, TES, HEPES, TAPSO, TAPS and the like. Thesebuffers are also commercially available.

Concentrations of respective reagents can be suitably selected. However,desirably, the concentration of thrombin in a final reaction mixture is0.01 to 10 NIHU/ml, preferably, 0.2 to 2 NIHU/ml. Desirably, theconcentration of the chromogenic substrate in a final reaction mixtureis 0.01 to 5 mM, preferably, 0.1 to 0.5 mM. Desirably, the concentrationof heparin in a mixture of a sample and thrombin is 0.02 to 100,000U/ml, preferably, 1 to 1,000 U/ml. Desirably, the salt concentration ina mixture of a sample and thrombin is more than 0.20M and up to 0.90M,preferably, 0.25M to 0.60M, more preferably, 0.35M to 0.60M. The pH's ofa mixture of a sample and thrombin and a final reaction mixture areadjusted with a buffer, desirably, in the range of 6.0 to 10.0,preferably, 7.5 to 8.5. Desirably, its concentration is 0.005 to 1.0M,preferably, 0.02 to 0.1M.

In the determination method of the present invention, various materialscan be suitably added to a thrombin reagent and a chromogenic substratereagent so as to maintain properties and quality of the reagents, tofacilitate the production thereof. Examples thereof include saccharides;amino acids; proteins such as albumin and the like; amine compounds suchas monoethylamine; and other materials such as polyethylene glycol,glycerol in addition to chelating reagents such as EDTA, EGTA;anti-fibrinolytic reagents such as epsilon aminocaproic acid, tranexamicacid, aprotinin; heparin inhibitors such as polybrene, protamine;preservatives such as sodium azide, gentamicin sulfate, thimerosal; andsurfactants such as Triton X-100, Tween 20.

An embodiment of the operation in the determination method of thepresent invention is illustrated hereinbelow.

Firstly, a sample and a thrombin reagent is mixed. Alternatively, asample may be mixed with a thrombin reagent containing heparin. In thisstage, a salt may be contained in either or both of thrombin reagent andreagent containing heparin or, in the case of a diluted sample, in asample.

The mixture is incubated at 15° to 50° C., preferably, 30° to 40° C. fora given time, e.g., 0.5 to 20 minutes, preferably, 1 to 5 minutes. Bythis incubation for a given time, ATIII in the sample is reacted withthrombin.

Then, a chromogenic substrate reagent is added and a color developedfrom the chromogenic substrate according to the remaining thrombinactivity is measured. The measurement is carried out either by a ratemeasurement wherein the change of absorbance per time is measured or byan end point measurement wherein a reaction stopping solution is addedafter a given period of time. By comparing the value measured with avalue obtained by using a standard sample, ATIII activity is determined.For the determination with an automatic analyzer, a rate measurement ispreferred. For an end point measurement, in general, 10 to 50% aceticacid or 10 to 20% citric acid solution is used as the reaction stoppingsolution. In the method of the present invention, 1 to 50 μl,preferably, 3 to 10 μl of a sample is used per 1 ml of a final reactionmixture.

Furthermore, the present invention provides an improved reagent kitwherein a salt is included in addition to a known reagent kit comprisingthrombin, heparin and a chromogen substrate in such an amount that thesalt concentration in a reaction mixture of a sample and thrombinbecomes more than 0.20M and up to 0.90M.

The reagent kit for the determination of ATIII activity of the presentinvention can be a mixture of constituent reagents or an assembly ofrespective constituent reagents. The mixture of constituent reagents orrespective constituent reagents can be in the form of a solution whereinthe constituent reagent(s) together with an excipient are dissolved indistilled water or a buffer solution in such a concentration that thedesired concentration in a reaction mixture can be obtained, and whichcan be directly served to use in the determination as it is, or in theform of a concentrated solution which is diluted to the desiredconcentration upon use, or in the form of a lyophilized productaccording to conventional methods. Among them, a lyophilized product isusually employed and, upon use, it is restored with distilled water or abuffer solution. Furthermore, respective constituent reagents can be inthe same forms or different forms.

In any case, the amount of a salt in the reagent kit is selected fromsuch a range that the salt concentration in a reaction mixture of asample and thrombin exceeds 0.20M and not more than 0.9M.

The following Reference Example and Examples further illustrate thepresent invention in detail.

REFERENCE EXAMPLE

The reference example was carried out according to the method disclosedin Example 1 of U.S. Pat. No. 4,918,001 (Apr. 17, 1990). To a specimen(20 μl ) was added 2 mM chromogenic substrate S-2238 (40 μl) and then tothe mixture was added a solution (200 μl) of 0.1M tris-HC1 buffercontaining thrombin (1 NIHU/ml) and heparin (2.5 U/ml) (thrombinreagent, pH 8.2). After 15 seconds up to 90 seconds, the change ofabsorbance at 405 nm was measured at 37° C. For measurement, anautomatic analyzer COBAS FARA II (manufactured by Roche, Switzerland)was used. Sodium chloride was added to the thrombin reagent in aconcentration of 0.1M, 0.2M, 0.3M, 0.4M or 0.5M. A calibration curve wasprepared by using a commercially available normal plasma CaliplasmaIndex 100 (manufactured by bioMerieux S.A., France) as a standard,physiological saline solution for diluting the specimen (ATIII activityof 25, 50 or 75%) and physiological saline solution itself as thespecimen for 0% of ATIII activity. In addition, 100% of ATIII activity(normal specimen) and 50% of ATIII activity (abnormal specimen) weremeasured 5 times at respective salt concentrations and ATIII activitieswere calculated from the calibration curve to evaluate simultaneousreproducibility.

As shown in FIG. 1, the calibration curve was prepared between sodiumchloride concentrations of 0.1M and 0.5M. However, the sensitivity atthe concentration of 0.5M was not preferred and the calibration curvewas not linear between the concentrations of 0.1M and 0.4M. Simultaneousreproducibility at sodium chloride concentration of 0.1M was 7.2 to12.7% of CV and the average CV of simultaneous reproducibilities ofrespective measurements at sodium chloride concentrations of 0.2 to 0.4Mwas 7.7%.

The calibration curve was not linear, reproducibility was as low asabout 7% and, as shown by FIG. 1 of U.S. Pat. No. 4,918,001 (Apr. 17,1990), the reaction time course was non-linear. This is therefore notdesirable for using as routine tests. Thus, it has been proved that theorder of operation is of importance.

EXAMPLE 1

Preparation of HCII fraction

Barium sulfate (10 g) was added to human normal plasma. (40 ml,purchased from Nippon Biological Material Center). After stirring themixture (for 15 minutes), it was centrifuged at 3,000 r.p.m. for 15minutes to separate a supernatant. The supernatant was passed through acolumn (plastic injection cylinder 35 ml manufactured by Nipro) ofheparin agarose equilibrated with 20 mM tris-HCl buffer (pH 7.3) (25 ml,Type II manufactured by Sigma, U.S.A.) to adsorb and remove ATIII. Theeffluent fraction was collected. Furthermore, for removing ATIIIcompletely, the recovered fraction was again passed through the samecolumn and the effluent fraction was collected. The effluent fractionwas concentrated to about 5 to 6 times with Centriprep-10® (manufacturedby Amicon, U.S.A.) to obtain HCII fraction.

EXAMPLE 2

Bovine thrombin (a constituent reagent of a fibrinogen kit manufacturedby bioMerieux S.A. of France) was dissolved with 50 mM tris-HC1 buffer(pH 8.4) containing 3 U/ml of heparin, 7.5 mM EDTA and 0.20 to 0.60Msodium chloride to bring the concentration to 3 NIHU/ml (thrombinreagent). A chromogenic substrate, 2AcOH-H-D-HHT-Ala-Arg-pNA (aconstituent reagent of a ATIII determination kit (ATIII Chrom)manufactured by bioMerieux S.A.) was dissolved with distilled water (1.4mM) to obtain a substrate reagent. A calibration curve was prepared byusing commercially available normal plasma, Caliplasma Index 100(manufactured by bioMerieux S.A.) and diluting with physiological salinesolution to obtain 0% (only physiological saline solution), 25%, 50% and100% (without dilution) of ATIII activities. By using the HCII fractionobtained in Example 1 as a specimen, the measurement was carried out andATIII activity was determined from the calibration curve to evaluate theinfluence of HCII. For the measurement, the automatic analyzer, COBASFARA, was used. In the determination method of the present invention,the thrombin reagent (200 μl) was added to the specimen (2 μl) and themixture was incubated at 37° C. for 3 minutes. Then, the substratereagent (100 μl) was added thereto and, from 20 to 90 seconds after theaddition, the absorbance at 405 nm was measured to determine the changeof absorbance per 1 minute.

As a result of the study of the influence of HCII on ATIIIdetermination, a great influence was observed at the salt concentrationof 0.20M which was employed in a conventional method, while no influenceof HCII was observed at a salt concentration of not less than 0.3M.

In addition, the influence of HCII was studied in more detail at sodiumchloride concentrations of 0.20 to 0.30M. The results are shown inTable 1. At the salt concentration of 0.22M, about 40% of the influencein a conventional method was avoided and, at the salt concentration of0.24M, about 60% influence avoidance effect was observed. At the saltconcentration of 0.26M, about 90% influence avoidance effect wasobserved and, at the salt concentration of 0.30M, no influence of HCIIwas observed. By adjusting the salt concentration of the reactionmixture of the sample and the thrombin reagent, HCII influence avoidanceeffect was observed. It has been found that ATIII can be determinedaccurately by utilizing this HCII influence avoidance effect.

                  TABLE 1                                                         ______________________________________                                        HCII Influence Avoidance Effect                                                            Concentration of NaCl                                                        M      0.20   0.22 0.24 0.26 0.28 0.30                            ______________________________________                                        ATIII activity (%)  36    22   14    4   2    0                               HCII influence (%) (100)  61   31   11   6    0                               ______________________________________                                    

EXAMPLE 3

According to the method of Example 2, the determination was carried outand a calibration curve was prepared with respect to the saltconcentration by using a thrombin reagent containing 2 NIHU/ml ofthrombin and using specimens for preparing the calibration curve havingATIII activities of 0, 25, 50, 75 and 100%. Furthermore, 100% of ATIIIactivity (normal specimen) and 50% (abnormal specimen) were measured 5times at the respective salt concentrations and ATIII activities weredetermined from the calibration curve to estimate simultaneousreproducibility. The calibration curve at the respective saltconcentrations is shown in FIG. 2.

At a salt concentration of less than 0.30M, in the case of 100% of ATIIIactivity, about 90% of thrombin added was inhibited and the linearity ofthe calibration curve was maintained at up to 75% of ATIII activity. Onthe other hand, at the salt concentration of 0.40M, inhibition ofthrombin was about 50% and, at the salt concentration of 0.50M,inhibition of thrombin was about 15%. The linearity was maintained up to100% of ATIII activity. However, under these conditions, desiredsensitivities were not obtained at 0.50M and 0.60M. The average CV ofsimultaneous reproducibilities of respective measurements at 0.20 to0.40M was 2.3%. This CV corresponds to the accuracy of measurementsuitable for using as routine tests.

In addition, according to the same method, proportions of remainingthrombin activities to 100% of ATIII activity were calculated at thesalt concentrations between 0.30M and 0.40M. The results are shown inTable 2.

As the salt concentration increases, the proportion of remainingthrombin activity increases.

These results show that more amount of a specimen can be used at a highsalt concentration and that the amount of thrombin activity to be addedas a reagent can be reduced.

                  TABLE 2                                                         ______________________________________                                        Salt Concentration and Proportion of Remaining Thrombin                                    Concentration of NaCl                                            ATIII       M      0.30   0.32 0.34 0.36 0.38 0.40                            ______________________________________                                        0% (ΔOD/min)                                                                           1.275  1.273  1.319                                                                              1.284                                                                              1.281                                                                              1.316                             100% (ΔOD/min)                                                                         0.096  0.176  0.280                                                                              0.344                                                                              0.593                                                                              0.689                             Remaining thrombin                                                                           7.5    13.8   21.2 26.8 46.3 52.3                              (%)                                                                           ______________________________________                                    

EXAMPLE 4

According to the method of Example 3, the relationship between saltconcentration and its calibration curve in the presence of a very highconcentration of heparin such as 1,000 U/ml was studied. The results areshown in FIG. 3. In view of these results and the results in Example 3,the salt concentration and heparin concentration have a relation to eachother and, in general, it is understood that a higher concentration ofheparin is required at a higher salt concentration, but a preferredheparin concentration is present with respect to a given saltconcentration. The results show that, even for a salt concentration ofhigher than 0.50M at which the sensitivity was not preferred in Example3, a salt concentration of 0.50 to 0.90M can be employed by suitablyselecting the concentration of heparin.

EXAMPLE 5

The ATIII activity determination kit of the present invention wasobtained by combining a thrombin reagent composed of 50 mM tris-HClbuffer solution (pH 8.4) containing 1.0 NIHU/ml of thrombin, 3 U/ml ofheparin, 7.5 mM EDTA and 0.4M sodium chloride with a substrate reagentcomposed of 1.4 mM chromogenic substrate, 2AcOH-H-D-HHT-Ala-Arg-pNA.

As described hereinbefore, according to the method of the presentinvention, the influence of HCII in ATIII determination can be avoidedby such a very simple method that a salt concentration in a reactionmixture of a conventional ATIII determination method is increased withminimum economical burden. The amount of thrombin to be used can bereduced and the method of the present invention can accurately, easilyand simply determine ATIII within a very short period of time withoutdilution of a sample and exceeding the measurable limit of absorbanceand is readily applicable to an automatic analyzer. Thus, the method ofthe present invention is useful for using as routine tests.

What is claimed is:
 1. A method for the determination of antithrombinIII activity which comprisesadding an excess amount of thrombin to asample, reacting antithrombin III in the sample with the thrombin in thepresence of heparin and a salt to form a reaction mixture, and thenmeasuring a remaining throbin activity in the reaction mixture with achromogenic substrate which develops a color by the reaction withthrombin, thereby indirectly determining the antithrombin III activityof the sample, the concentration of said salt in the reaction mixturebeing more than 0.20M to not more than 0.90M.
 2. A reagent kit for thedetermination of antithrombin III activity without dilution of a sample,said kit comprising (a) a thrombin reagent containing heparin, and asalt, and (b) a chromogenic substrate reagent, the amount of the salt in(a) being such that the salt concentration in a reaction mixture of apredetermined volume of sample and a predetermined volume of thethrombin reagent becomes more than 0.20M and up to 0.90M.
 3. A reagentkit for the determination of antithrobin III activity with dilution of asample which comprises:(a) a sample-dilution solution, (b) a thrombinreagent, and (c) a chromogenic substrate reagent, wherein heparin and asalt are contained in the sample-dilution solution or the thrombinreagent or both the sample-dilution solution and the thrombin reagent,the amount of the salt being such that the concentration of said salt ina mixture of respective predetermined volumes of the sample, thesample-dilution solution, and the thrombin reagent becomes more than0.20M and up to 0.90M upon formation of said mixture.
 4. A methodaccording to claim 1, wherein the concentration of said salt is not lessthan 0.25M to 0.60M.
 5. A method according to claim 1, wherein theconcentration of said salt is not less than 0.35M to 0.60M.
 6. A methodaccording to claim 1, wherein the sample comprises plasma.
 7. A methodaccording to claim 1, wherein the salt is selected from the groupconsisting of sodium chloride and potassium chloride.
 8. A methodaccording to claim 1, wherein the sample is not diluted prior to addingthe thrombin to the sample.
 9. A method according to claim 1, whereinthe concentration of thrombin in the reaction mixture is 0.01 to 10NIHU/ml.
 10. A method according to claim 1, wherein the concentration ofthe chromogenic substrate reagent in the reaction mixture is 0.01 to 5mM.
 11. A method according to claim 1, wherein the concentration ofheparin in the reaction mixture is 0.02 to 100,000 U/ml.
 12. A methodaccording to claim 1, wherein the pH of the reaction mixture is in arange of 6.0 to 10.0.
 13. A method according to claim 1, wherein thethrombin added to the sample also includes heparin and the salt.
 14. Amethod according to claim 1, wherein the heparin, and salt are addedafter the thrombin is added to the sample.